This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin and/or rapamycin metabolites, derivatives and analogues. The reactivity of these polyclonal and monoclonal antibodies makes them particularly useful for immunoassays for therapeutic drug monitoring (TDM). These immunoassays or TDM kits may include polyclonal or monoclonal antibodies to specific sites of Rapamycin (Rapa) and/or metabolites, derivatives and analogues of rapamycin. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies.
This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of rapamycin (Sirolimus). The reactivity of these poly and monoclonal antibodies make them particularly useful for immunoassays for therapeutic drug monitoring (TDM). These immunoassays or TDM kits may include polyclonal or monoclonal antibodies to specific sites of rapamycin. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies.
Rapamycin (Rapa) is a macrocyclic antibiotic (macrolide lactone), which was originally isolated in soil samples from Easter Island from a Streptomyces hygroscopicus strain1. Rapamycin is structurally related to the immunosuppressant FK-506 (Tacrolimus) but mechanistically different. Rapamycin has anti-candidal, anti-proliferative and anti-tumor activity. Rapamycin also dampens autoimmune reactions (SLE, adjuvant arthritis, allergic encephalomyelitis). Rapamycin is also a potent immunosuppressant that inhibits T and B cell activation by blocking cytokine-mediated events, and inhibits growth factor mediated cell proliferation. The structure of rapamycin is given in FIG. 1.
Currently, the two most commonly administered immunosuppressive drugs to prevent organ rejection in transplant patients are Cyclosporine (CSA) and FK-506 (FK). Therapeutic monitoring of concentrations of these drugs in blood is required to optimize dosing regimes to ensure maximal immunosuppression with minimal toxicity. Recent clinical data indicates that Rapamycin will be a widely used immunosuppressant to prevent organ rejection in transplant patients. Specific TDM monitoring kits for Rapamycin will therefore be required. The polyclonal and monoclonal antibodies to specific sites of Rapamycin of this invention are ideally suited for developing Rapamycin TDM kits.
Cytochrome P4503A4 enzyme metabolizes rapamycin to a number of demethylated and hydroxylated metabolites. The exact pathways of rapamycin metabolism in humans have not been completely elucidated since only a few of the metabolites have been structurally identified. Therefore, no consensus has been established concerning the identity or steady state concentrations in whole blood after oral administration. A summary of the current reported knowledge of rapamycin metabolism follows.
Streit et. al. structurally identified four rapamycin metabolites from rabbit liver microsomes2. These include 41-demethyl rapamycin, 7-demethyl rapamycin, 11-hydroxy rapamycin, and a 24-hydroxy ester hydrolysis degradation product of rapamycin. It has also been shown that the metabolites of rapamycin can undergo this ester hydrolysis. Streit also partially identified di, tri, and tetra hydroxylated rapamycin metabolites. Wang et. al. found 16 hydroxylated and/or demethylated metabolites in the bile of rapamycin treated rats3. Nickmilder et. al. identified a 3,4 and 5,6 dihydrodiol rapamycin metabolite in rat liver microsomes.4 In trough whole blood, Streit et. al. have identified 41-demethyl, dihydroxy, and didemethyl rapamycin metabolites.5 These metabolites accounted for 56% of total rapamycin derivatives measured. Finally, Leung et. al. looked at the disposition of [14C]-rapamycin in healthy male volunteers.6 They found that rapamycin represented approximately 35% of the total radioactivity in blood and that 41-demethyl, 7-demethyl, and several hydroxy, hydroxydemethyl, and didemethyl rapamycin metabolites individually represented between 1 and 12% of the total radioactivity. They also found there was no notable presence of glucuronide or sulfate conjugates in blood, feces, or urine and that most of an oral dose was eliminated in feces. Rapamycin metabolites can be isolated from a number of various sources, including but not limited to blood, urine or feces samples, from liver microsomes or from microorganism cultures.
There is a need for improved methods of monitoring levels of rapamycin and/or rapamycin metabolites and derivatives.
The current invention is drawn to methods for the preparation of immunogenic conjugates which elicit antibodies with specificity for rapamycin related compounds. For the purposes of this application, the term rapamycin related compound is meant to include any or all of the rapamycin molecule itself and/or various rapamycin metabolites and derivatives. Rapamycin and rapamycin metabolite and/or derivative conjugate immunogens are prepared and used for the immunization of a host animal to produce antibodies directed against specific regions of the rapamycin or metabolite and/or derivative molecules. By determining the specific binding region of a particular antibody, immunoassays which are capable of distinguishing between the parent molecule, active metabolites, inactive metabolites and other rapamycin derivatives/analogues are developed. The use of divinyl sulfone (DVS) as the linker arm molecule for forming rapamycin/metabolite/derivative-protein conjugate immunogen is described.
In a first aspect, the invention provides antibodies which are capable of binding to a rapamycin related compound. Such antibodies which recognize a specific region of said rapamycin related compound, the Rapa derivative RAD or the Rapa metabolites M1 to M5 are preferred. Monoclonal antibodies (MoAbs) are most preferred. Also provided are methods for producing an antibody which is capable of recognizing a specific region of rapamycin related compound, said methods comprising: a) administering an immunogen comprising a rapamycin related compound, a linker arm molecule and a protein carrier to an animal so as to effect a specific immunogenic response to the rapamycin related compound; b) recovering an antibody to said rapamycin related compound from said animal; and c) identifying the antibody binding region by measuring the reactivity of the antibody to at least one rapamycin related compound. Such methods wherein said linker arm molecule is divinyl sulfone and where the rapamycin related compound is linked to the carrier at the 27, 31, 41 or 42 position are preferred. The protein carrier may preferably be keyhole limpet hemocyanin or human serum albumin. Use of hybridoma cells to accomplish the above methods is also provided.
In another aspect, the invention provides immunoassay methods for measuring the level of a rapamycin related compound in a mammal, comprising: a) incubating a biological sample from said mammal with at least one antibody which is capable of binding to a rapamycin related compound; and b) measuring the binding of rapamycin related compound to said antibody. Use of antibodies which recognize a specific region of said rapamycin related compound, the Rapa derivative RAD or the Rapa metabolites M1 to M5 in these assays is preferred. Use of monoclonal antibodies is most preferred. Immunoassay kits for measuring the level of a rapamycin related compound in a sample, said kits comprising an antibody as described above are also provided.